WO2015053431A1 - Procédé de préparation d'allinase dérivé de l'ail - Google Patents

Procédé de préparation d'allinase dérivé de l'ail Download PDF

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Publication number
WO2015053431A1
WO2015053431A1 PCT/KR2013/011169 KR2013011169W WO2015053431A1 WO 2015053431 A1 WO2015053431 A1 WO 2015053431A1 KR 2013011169 W KR2013011169 W KR 2013011169W WO 2015053431 A1 WO2015053431 A1 WO 2015053431A1
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WIPO (PCT)
Prior art keywords
garlic
buffer
derived
alinase
minutes
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PCT/KR2013/011169
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English (en)
Korean (ko)
Inventor
이영춘
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(주)태영에프에이
이영춘
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Application filed by (주)태영에프에이, 이영춘 filed Critical (주)태영에프에이
Publication of WO2015053431A1 publication Critical patent/WO2015053431A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y404/00—Carbon-sulfur lyases (4.4)
    • C12Y404/01—Carbon-sulfur lyases (4.4.1)
    • C12Y404/01004—Alliin lyase (4.4.1.4)

Definitions

  • the present invention relates to a method for preparing garlic-derived alinases.
  • Garlic ( Allium sativum L.) is a representative Allium ( Allium ) plant belonging to the family Liliaceae has a unique flavor and various biological activities. Garlic has been used as a nutritious food, tonic, spices, etc., and contains more energy, vitamins and minerals than other vegetables, and is rich in phosphorus, potassium, germanium, vitamin B1, and vitamin B2. Garlic not only enhances the taste of food, but also has food preservation ability, and antibacterial action that inhibits the growth of bacteria such as food poisoning bacteria, antihypertensive and blood pressure lowering effect, anticancer effect, and antimutagenic effect of cells are known. Accordingly, in Korea, garlic is used in many forms such as garlic pickles, kimchi or stew, soup, herbs, and radish, or it is dried and powdered to be used as a compound seasoning material.
  • Alyinase is an enzyme present in leeks, garlic, and leeks. It is weak in acid and heat. When garlic is ground, allin is broken down by the action of an allinase enzyme and two molecules are condensed into allicin. Allicin with disulfide bonds is well decomposed to heat like other disulfide compounds. The spicy taste and garlic's distinctive aroma that comes from tearing or crushing garlic when eating fresh garlic or cooking is due to sulfur compounds, including allicin. Since allicin is a kind of phytoncide, it has antibacterial and antifungal properties.
  • Korean Patent Publication No. 10-1084316 describes a method of preparing garlic seasoning material by high pressure / enzymatic digestion process.
  • studies on how to separate aliinase from garlic and use it are insignificant.
  • the inventors of the present invention continued the study on the preparation method for separating aliinaase from garlic, confirming that the ratio of garlic to acid or alkaline buffer, pH, and the number of extractions affect the yield of aliinaase. Was completed.
  • Still another object of the present invention is to provide a garlic-derived alinase prepared by the above method.
  • the present invention provides a method for producing garlic-derived alinases.
  • the present invention provides a garlic-derived kinases produced by the above production method.
  • Garlic-derived alinases according to the present invention can be usefully used in foods and the like as a method to maximize the yield of alinases.
  • step b) adding an acidic buffer to the liquid fraction obtained in step a) to adjust the pH to 4.5 to 5.5 and then centrifuging to obtain a solid;
  • step b) adding an alkaline buffer to the solids obtained in step b) to adjust the pH to 6 to 8, and then adding a weighting agent to obtain an allinase;
  • It provides a method of producing a garlic-derived kinases, comprising; d) powdering the frozen lyase obtained in step c) after freezing and drying.
  • the present invention provides a garlic-derived kinases produced by the above production method.
  • the aliinase according to the present invention was prepared by adding acidic or alkaline buffer to garlic to adjust the pH to obtain a liquid fraction, and then adding an acidic buffer to adjust the pH to 4.5 to 5.5 to obtain a solid, wherein the alkaline buffer After adjusting the pH to 6 to 8 to add a weighting agent to obtain alinain, it is characterized in that it is frozen, dried and powdered.
  • Step a) is a step of obtaining a liquid fraction of garlic, the pH of the pH of 4 or less by mixing acidic or alkaline buffer with garlic in a volume ratio of 1: 2 to 10, preferably 1: 5 to 7 After adjusting to 7 or more, preferably 2 to 4 or 7 to 10, centrifugation yields a liquid fraction.
  • the volume ratio of garlic to buffer is 1: 2 or less, it is difficult to obtain a liquid fraction.
  • the volume ratio is 1:10 or more, the increase in yield of the liquid fraction is not remarkable.
  • the centrifugation conditions are 10 to 20 minutes at 10,000 to 12,000 rpm, preferably 15 minutes at 10,000 rpm. At this time, when centrifugation at less than 10,000rpm, it is difficult to separate the insoluble material, and the degree of suspended sediment is high, so that the recovery rate of aliinaase is low.
  • the acidic buffers include, but are not limited to, phosphate buffers, acetate buffers, citrate buffers and formate buffers.
  • the alkaline buffers include but are not limited to sodium hydroxide buffer, ammonium hydroxide buffer, potassium hydroxide buffer and calcium hydroxide buffer.
  • Step b) is a step of obtaining a solid from the liquid fraction of garlic, the acidic buffer is added to the liquid fraction obtained in step a) again to adjust the pH to 4.5 ⁇ 5.5, preferably pH 4.9 and centrifuged Obtain solids.
  • the centrifugation conditions are preferably made the same as the conditions performed in step a).
  • the step c) is to obtain the alinase, the alkaline buffer solution is added to the solid obtained in the step b) to adjust the pH to 6 to 8, preferably pH 7, and then add a weighting agent to the alinase Obtain alliase.
  • the weight agent may be one or more selected from the group consisting of sugars, polysaccharides, dextrins, celluloses, synthetic polymers, semisynthetic polymers, amino acids, polyamino acids, proteins and lipids, preferably dextrin And more preferably maltodextrin.
  • Step d) is a step of freezing and drying the powder after the aliinaase obtained in step c).
  • the freezing may be performed at -15 ° C to -30 ° C, preferably at -20 ° C.
  • the drying may be performed by natural drying, hot air drying, cold air drying, vacuum drying, far-infrared drying, high frequency drying, ultrasonic drying, foam drying, drum drying, puffed drying method, but preferably 1 to 3 torr and 40 to It may be carried out at 60 °C, more preferably at 1 torr and 45 °C.
  • the process can be repeated extracting 1-2 times to obtain a linase.
  • the optimum conditions of the garlic-derived alinase preparation method according to the present invention is the mixing ratio of garlic to buffer is 1: 2 ⁇ 10 volume ratio, the centrifugation conditions are 10-20 minutes at 10,000 ⁇ 12,000rpm When the pH of the solid is 4.5-5.5, the yield of alinaase is best.
  • the resulting aliinaase was frozen at -20 ° C, dried at 45 ° C at a pressure of 1 torr, and then ground to powder. Extraction was repeated 1-2 times with the preparation method to finally prepare a garlic-derived alinase.
  • the centrifugation conditions for 10 to 20 minutes at 10,000 to 12,000 rpm can effectively separate insoluble materials and increase the recovery rate of alinase due to less flotation of sediment. Confirmed.
  • the pH range was set in consideration of the pH of the solubility of the alinaase.
  • 0.1M phosphate buffer was added to the liquid fraction obtained by centrifugation in Experimental Example 2 to suit each pH condition, and left to stand for 30 minutes or more, and centrifuged again under the optimum conditions of Experimental Example 2 to obtain a solid.
  • the obtained solids were frozen at -20 ° C and then dried in a freeze dryer (Ishinshin Bio) at 1 torr pressure and 45 ° C, and weighed to determine the recovery rate of alinase.
  • the recovery rate of Alinaase according to pH is shown in Table 3 below.
  • the alinase recovery was the lowest value at 3.6% at pH 4.6, and the Alinaase recovery was highest at 4.5% when the pH was adjusted to 4.9. There was a difference in percent recovery. Therefore, it was confirmed that precipitation of the liquid fractions by adjusting the pH to 4.9 can increase the recovery rate of alinase.
  • the garlic was dried and simply powdered as a control group, and the extract extracted by the method of Experimental Examples 1 to 4 was extracted once, and the once-dried non-dried solid was dissolved at pH 8 and precipitated at pH 4.9 after centrifugation.
  • the powdered product was subjected to twice extraction.
  • the activity and yield of the powdered garlic, aliinase obtained by extracting once and twice were measured. 0.5 ml aliinase and 1 ml allin reaction solution were mixed and reacted at 25 ° C. for 5 minutes. 1.5 ml of 10% TCA was added to the reaction, followed by centrifugation at 10,000 rpm for 15 minutes. 0.1% 2,4DNPH was added to the centrifuged supernatant, and then reacted at 25 ° C. for 5 minutes, and 5 ml of 0.5 mol NaOH was added thereto and reacted at 25 ° C. for 5 minutes. Alinase activity was measured by measuring absorbance using a spectrophotometer (Beckman Co.) at 515 nm conditions.
  • the alinase activity was 414.9 unit / g in the powdered garlic, and the extract showed twice the activity of 865.7 unit / g, 2.1 times higher than the powdered garlic, and twice the extraction. Showed an activity of 925.5 units / g, which was 2.23 times higher than the one extraction.
  • the yield of allinaase was the highest at 4.5% in one extraction. Therefore, the increase in the number of repeated extraction increased the alinase activity but the yield was slightly reduced, it was confirmed that the optimal number of extraction is 1-2 times.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Preparation Of Fruits And Vegetables (AREA)

Abstract

L'invention concerne un procédé de préparation d'allinase dérivé de l'ail. Le procédé de l'invention permet d'optimiser le rendement de l'allinase, et peut ainsi s'utiliser pour des aliments, etc.
PCT/KR2013/011169 2013-10-08 2013-12-04 Procédé de préparation d'allinase dérivé de l'ail WO2015053431A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20130119943A KR20150041425A (ko) 2013-10-08 2013-10-08 마늘 유래 알리이나아제의 제조방법
KR10-2013-0119943 2013-10-08

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WO2015053431A1 true WO2015053431A1 (fr) 2015-04-16

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WO (1) WO2015053431A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114478331A (zh) * 2022-02-17 2022-05-13 齐鲁工业大学 一种蒜氨酸的分离纯化方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994008614A1 (fr) * 1992-10-08 1994-04-28 Yeda Research And Development Co. Ltd. Alliinase de recombinaison, sa preparation et compositions pharmaceutiques la contenant
JP2000508535A (ja) * 1996-04-16 2000-07-11 イエダ リサーチ アンド デベロップメント カンパニー リミテッド 固定化アリイナーゼおよびアリシンの連続生産
CN1424397A (zh) * 2003-01-13 2003-06-18 陈坚 从鲜蒜中提取蒜酶生产工艺
CN101191126A (zh) * 2006-11-28 2008-06-04 河南农业大学 一种蒜氨酸酶的一步分离纯化方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994008614A1 (fr) * 1992-10-08 1994-04-28 Yeda Research And Development Co. Ltd. Alliinase de recombinaison, sa preparation et compositions pharmaceutiques la contenant
JP2000508535A (ja) * 1996-04-16 2000-07-11 イエダ リサーチ アンド デベロップメント カンパニー リミテッド 固定化アリイナーゼおよびアリシンの連続生産
CN1424397A (zh) * 2003-01-13 2003-06-18 陈坚 从鲜蒜中提取蒜酶生产工艺
CN101191126A (zh) * 2006-11-28 2008-06-04 河南农业大学 一种蒜氨酸酶的一步分离纯化方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RABINKOV, AARON ET AL.: "Alliinase (alliin lyase) from garlic (Alliium sativum) is glycosylated at ASN146 and forms a complex with a garlic mannose-specific lectin", GLYCOCONJUGATE JOURNAL, vol. 12, no. 5, 1995, pages 690 - 698 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114478331A (zh) * 2022-02-17 2022-05-13 齐鲁工业大学 一种蒜氨酸的分离纯化方法
CN114478331B (zh) * 2022-02-17 2023-08-11 齐鲁工业大学 一种蒜氨酸的分离纯化方法

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